Rho Kinase as a Target
The Rho family of small GTP binding proteins can be activated by several extracellular stimuli such as growth factors, hormones and mechanic stress and function as a molecular signaling switch by cycling between an inactive GDP-bound form and an active GTP-bound form to elicit cellular responses. Rho kinase (ROCK) functions as a key downstream mediator of Rho and exists as two isoforms (ROCK 1 and ROCK 2) that are ubiquitously expressed. ROCKs are serine/threonine kinases that regulate the function of a number of substrates including cytoskeletal proteins such as adducin, moesin, Na+-H+ exchanger 1 (NHE1), LIM-kinase and vimentin, contractile proteins such as the myosin light chain phosphatase binding subunit (MYPT-1), CPI-17, myosin light chain and calponin, microtubule associated proteins such as Tau and MAP-2, neuronal growth cone associate proteins such as CRMP-2, signaling proteins such as PTEN and transcription factors such as serum response factor (Loirand et al, Circ Res 98:322-334 (2006)). ROCK is also required for cellular transformation induced by RhoA. As a key intermediary of multiple signaling pathways, ROCK regulates a diverse array of cellular phenomena including cytoskeletal rearrangement, actin stress fiber formation, proliferation, chemotaxis, cytokinesis, cytokine and chemokine secretion, endothelial or epithelial cell junction integrity, apoptosis, transcriptional activation and smooth muscle contraction. As a result of these cellular actions, ROCK regulates physiologic processes such as vasoconstriction, bronchoconstriction, tissue remodeling, inflammation, edema, platelet aggregation and proliferative disorders.
One well documented example of ROCK activity is in smooth muscle contraction. In smooth muscle cells ROCK mediates calcium sensitization and smooth muscle contraction. Agonists (noradrenaline, acetylcholine, endothelin, etc.) that bind to G protein coupled receptors produce contraction by increasing both the cytosolic Ca2+ concentration and the Ca2+ sensitivity of the contractile apparatus. The Ca2+-sensitizing effect of smooth muscle constricting agents is ascribed to ROCK-mediated phosphorylation of MYPT-1, the regulatory subunit of myosin light chain phosphatase (MLCP), which inhibits the activity of MLCP resulting in enhanced phosphorylation of the myosin light chain and smooth muscle contraction (WO 2005/003101A2, WO 2005/034866A2).
The use of prototype non-potent Rho-kinase inhibitors, Y27632 or fasudil, in animal models has suggested a number of potential benefits of Rho-kinase inhibitors, Y27632 has shown favorable activity in animal models of neoplasias and tumor metastasis (Ogawa T Am J Transplantation 7:347-355, 2007). Fasudil has been shown to have favorable activity in models of tumor growth and metastasis (Ying H et al. Mol Cancer Ther 5:2158-64, 2006). In addition, fasudil has been shown to provide benefits for controlling cerebral vasospasms and ischemia following subarachnoid hemorrhage in humans.
Diabetic Nephropathy
Diabetic Nephropathy (also known as Kimmelstiel-Wilson disease and intercapillary glomerulosclerosis) is kidney disease or damage that results as a complication of diabetes. It is the leading cause of death and disability in diabetes, and is characterized by nephrotic syndrome and nodular glomerulosclerosis. (Adler et al., Secondary Glomerular Diseases, in The Kidney. 5th, BM Brenner (ed). Pages 1498-1596). Diabetic nephropathy complicates approximately 20-30% of type 1 and type 2 diabetes mellitus and is clinically diagnosed by proteinuria and progressive renal insufficiency. Diabetic nephropathy is the leading cause of end-stage renal diseases in western societies and accounts for 30-35% of patients on renal replacement therapy. The exact cause of diabetic nephropathy is unknown, but it is believed that uncontrolled high blood sugar leads to the development of kidney damage. Diabetic nephropathy is a prime cause for dialysis in many Western countries (Parving et al., Diabetic Nephropathy, in The Kidney. 5th ed. B M Brenner (ed) Pages 1864-1892. (1996)).
A growing body of evidence suggests that the RhoA/ROCK pathway may have modulatory effects on the renal system. RhoA/ROCK pathway has been shown to play an important role in renal fibrosis by enhancing signaling pathways involving transforming growth factor-β, angiotensin II, and nuclear factor-B (Heusinger-Ribeiro, et al., J Am Soc Nephrol 12:1853-1861 (2001); Sharpe, et al., J Am Soc Nephrol 14:261-264 (2003)) as well as in VEGF-induced glomerular endothelial cell hyperpermeability (Kolavennu, et al., Diabetes 57:714-723 (2008)). Fasudil and Y-27632, two selective ROCK inhibitors not of Formula I or II, have been shown to improve outcomes in a variety of renal injury models in animals, including unilateral ureteral obstruction (Nagatoya, et al., Kidney Int 61:1684-1695 (2002)), hypertensive glomerulosclerosis (Kanda, et al., Kidney Int 64:2009-2019 (2003)) and ischemia/reperfusion-induced acute renal failure (Teraishi et al., Eur J Pharmacol 505:205-211, (2004)).
Optimization of glycemic control and inhibition of the renin-angiotensin system (RAS) are the mainstays of management of diabetic nephropathy, but renal damage progresses in many patients despite these measures (Bach, Diabetes 57:532-3 (2008)). Improved approaches to treating nephropathy are therefore required.
Malaria
Malaria currently represents one of the most prevalent infections in tropical and subtropical areas throughout the world. Per year, malaria infections kill up to 2.7 million people in developing and emerging countries. Malaria is caused by mosquito-borne hematoprotozoan parasites belonging to the genus Plasmodium. There are four species of Plasmodium protozoa (P. falciparum, P. vivax, P. ovale and P. malariae) that are responsible for the disease in man, The symptoms of uncomplicated malaria include fever, malaise, fatigue, muscle aches, back pain, headache, dizziness, loss of appetite, nausea, vomiting, abdominal pain, and diarrhea. Malaria may cause anemia and jaundice due to the destruction of red blood cells. P. falciparum accounts for the majority of infections in humans and is the most lethal type because the disease can progress to cerebral malaria. Cerebral malaria mostly affects young children and pregnant women in sub-saharan Africa, whereas multi-organ failure is seen mainly in adults in South East Asia. Severe malaria cases may then progress rapidly, often within 24 hours, to coma and death if left untreated. The current standard of treatment is with antimalarials or quinine. Antimalarials resistance has become a significant problem, and while combination therapies and quinine treatments offer some solution, combination therapies are often more expensive while quinine has greater side effect risks such as causing birth defects in pregnant women. The complex life cycle of the parasite and its constantly changing antigens have thus far prohibited vaccine development (Greenwood B et al. J Clin Invest, 118(4): 1266-1276, 2008). Therefore, there is a significant need for novel malarial therapies.
Endothelial activation is believed to play a crucial role in P. falciparum malaria acute phase severity. P. falciparum cytoadherence is known to induce proinflammatory endothelial responses via NF-kappaB signaling cascades. In addition, adherence of parasitized cells or of parasites at the surface of uninfected target endothelial cells is known to induce the activation of Rho kinase through membrane borne adhesion molecules. Members of the Rho family of small GTPases are known to play a pivotal role in the signal transmission of various receptors, including ICAM-1, vascular cell adhesion molecule (VCAM)-1 and selectins involved in cytoadherence and in the formation of focal adhesions (Taoufiq Z et al. J. Infectious Diseases, 197:1062-1073, 2008). In endothelial cells adhered by infected erythrocytes, activation of Rho kinase leads to Akt kinase inhibition and apoptosis of the endothelial cells and these effect are inhibited by fasudil, a known Rho kinase inhibitor compound not of Formula I or II.
Cancer
Cancer is the second most common cause of death in the US, exceeded only by heart disease. In the US, cancer accounts for 1 of every 4 deaths. This year, about 565,650 Americans are expected to die of cancer, more than 1,500 people a day. Cancer is a term that identifies a group of diseases characterized by uncontrolled growth of abnormal cells that are able to invade other tissues. In cancer patients, malignant tumors undergo extensive invasion and metastasis, the extent of which negatively impacts their prognosis and benefit from therapy. Metastasis of tumors and complications thereof, are the primary cause for cancer mortality. There is a great need to identify molecular pathways that are necessary for tumor invasion and metastasis and develop pharmacological agents that could effectively inhibit or block these processes (Ying H et al. Mol Cancer Ther, 5:2158-2164, 2006).
The ability of cancer cells to undergo invasion and migration is a prerequisite for tumor metastasis. The effects of a representative Rho kinase inhibitor compound, Y-27632, not of Formula I or II on the in vitro invasion of LPA-stimulated MM1 cells are to inhibit the migration of MM1 cells in a concentration dependent manner. The relative potencies of these compounds for inhibition of invasion were consistent with their inhibition constants (Ki) for inhibition of the kinase activity of ROCK in vitro (Itoh K et al. Nature Medicine, 5:221-5, 1999). Similarly, fasudil, another Rho kinase inhibitor compound not of Formula I or II has been shown to inhibit in vivo tumor progression in rodent model via alterations in cell migration and cell growth (Ying H et al. Mol Cancer Ther, 5:2158-2164, 2006).
There is a need for an effective or improved method for treating diseases or conditions associated with alteration in cellular integrity, permeability, proliferation or remodeling such as diabetic nephropathy, malaria, and cancer.